The present invention is directed to nucleic acid sequences encoding a limbic-system associated membrane protein (xe2x80x9cLAMPxe2x80x9d) and to purified proteins with LAMP activity. LAMP is a self-binding, antibody-like cell surface adhesion protein, the presence of which on one neuron of the limbic system stimulates the formation of connections with adjacent neurons.
A monoclonal antibody, 2G9, that bound to the cell surface of certain neurons, mostly limbic system or limbic system-connected neurons had been identified. Levitt, Science 223:299-301, 1984. This antibody has been confirmed to be specific for LAMP. Explanted neurons of the septum express the 2G9 antigen and, in culture, normally will invade explanted tissue from the hippocampus. In 1989, it was reported that 2G9 blocked that invasive process. Keller et al., Neuron 3:551-561, 1989. Efforts were made to clone the antigen using 2G9. In 1989, these efforts lead to the submission to the Society of Neuroscience of an erroneous abstract reporting that the antigen had been cloned. Efforts to clone the antigen using the antibody have proved unsuccessful.
Immunochemical studies indicate that the antigen is a 64-68 kDa glycoprotein that is expressed by cortical and subcortical neurons of the limbic system. These brain areas form functional circuits involved in memory, learning, mood, affect, cognitive behavior and central autonomic regulation. The antigen is also expressed by neurons having interconnections with limbic brain areas. Early in development, the antigen is expressed on limbic-related neurons. During pathway formation and differentiation, the antigen is expressed transiently on neural growth cones and axons.
The immunochemistry data indicate that the antigen, now designated LAMP, is a cell-surface adhesion molecule involved in directing the growth and differentiation of certain neurons. LAMP, when purified using the monoclonal antibody and coated onto tissue culture plates, induces adhesion and the growth of neuritic processes of certain limbic neurons, but generally not of non-limbic neurons.
Despite sustained efforts to isolate the gene for the 2G9 antigen, it is only now that the nucleic acid encoding LAMP has been identified. It is herein designated SEQ ID NO: 1 for the human LAMP (SEQ ID NO:2) and SEQ ID NO: 3 for the entire protein-encoding rat sequence (SEQ ID NO:41). The portions of SEQ ID NO: 3 encompassing only the open reading frame are designated SEQ ID NO: 7. These sequences encode all or part of polypeptides of 338 amino acids and indicate that the protein is highly conserved, since the human and rat sequences differ in only four amino acid residues (approximately 99% homologous).
As indicated in FIG. 2, the sequence data indicate that LAMP has a three domain immunoglobin structure. Studies with phosphatidyl inositol (xe2x80x9cPIxe2x80x9d) specific phospholipase C indicate that LAMP is bound to the cell membrane via a PI linkage. Thus, LAMP is much like other adhesion molecules of the immunoglobin superfamily that are attached to the cell surface by a PI linkage, such as TAG-1, Thy-1 and a form of NCAM. LAMP differs from these adhesion molecules in the specificity of its target tissues. The immunoglobulin domains of LAMP make up the binding or self-adhesion domain of LAMP.
In a first embodiment, the invention relates to a nucleic acid having a sequence encoding all of or a fragment of a LAMP effective to bind to a native LAMP at the cell surface of a neuron. In a further preferred embodiment, the nucleic acid encodes a water-soluble fragment of a LAMP. In still another preferred embodiment, the nucleic acid is a full-length-LAMP-encoding nucleic acid.
In yet another embodiment, the invention provides a nucleic acid encoding a mutant of the LAMP or fragment of the first embodiment.
In a further embodiment, the invention relates to a fragment of LAMP or a mutated LAMP fragment effective to bind to a native LAMP at the cell surface of a neuron to inhibit or stimulate the biological activity of the native LAMP.
In other embodiments, the invention relates to methods of treating disease, methods of diagnosing a developmental, psychotic or affective disorder, methods for detecting and measuring LAMP, nucleic acid polymerase chain reaction (xe2x80x9cPCRxe2x80x9d) primers, tissues histochemically stained using the nucleic acids of the invention, cells transformed with the nucleic acids of the invention, vectors comprising the nucleic acids of the invention (in sense or antisense orientation), methods of transforming stem cells to promote differentiation to limbic system neurons or neurons forming connections to limbic system neurons, methods of using such transformed stem cells to treat a neurological disease, reagents for targeting a biological agent to the limbic system, nucleic acids for creating cell lines and transgenic animals for testing biological agents for their ability to interfere with the activity of LAMP, methods of targeting limbic cells for gene therapy by functionally attaching the gene sought to be expressed to a LAMP promoter (see SEQ ID NO: 5 and FIG. 5), and nucleic acids defining cell-type specific LAMP promoters.
Still another embodiment of the invention uses the nucleic acid of the invention, including the PCR primers, to probe for LAMP-gene polymorphisms associated with diseases that affect the limbic system. These diseases include, without limitation, schizophrenia, familial epilepsy, manic-depression, obsessive-compulsive disorder, mood disorders and other affective psychiatric disorders.